The MSG1 family of transcriptional activators (MSG1, MRG1, nad SPECK) are small nuclear proteins that share two conserved regions, CR1 and CR2; the latter is necessary and sufficient for their strong transcriptional activating activity. Since they apparently lack DNA- binding activity, we hypothesize that they may interact with sequence- specific DNA-binding proteins and function as "transactivating subunits" of multi-subunit transcription factors. Supporting this hypothesis, we recently have discovered that MSG1 enhances transcriptional activation mediated by the Smad family signal transducer/DNA-binding proteins in a manner dependent of TGFbeta signaling. In this grant, we propose to elucidate the molecular mechanisms of this activity of MSG1. For this purpose, Msg1-deficient embryonic fibroblasts will be prepared from Msg1-mutant knockout mice, which we have already generated, and the enhancing effect of MSG1 on Smads-mediated transcriptional activation will be characterized using them by transfection-based analyses. We will also characterize expected physical interactions of MSG1 with the SMAD proteins and components of the transcription initiation complex in vitro using purified proteins and biochemical analyses, such as immunoprecipitation or electromobility shift assay. In vivo complex formation of MSG1 with such proteins will be evaluated by biochemical analyses of plasmid-derived proteins or endogenous proteins. To understand the physiological properties of MSG1, we propose to characterize the phenotypes of the Msg1-deficient mice and their embryonic fibroblasts, with genetic backgrounds of wild type or heterozygous mutations of Smad2 or Smad4. We will also characterize molecular mechanisms of MSG1-induced aggregation of B16-F10 melanoma cells, attempting to identify target gene(s) of MSG1-enhanced transcriptional activation. Elucidation of the physiological properties and the molecular mechanisms of action of MSG1 will provide insights as to how the MSG1 family proteins function as well as how the Smad2- mediated transcription is regulated by non-Smad proteins.